This invention relates generally to nucleic acid hybridization analysis. More specifically, a method for detecting a point mutation in a DNA strand is provided, which method uses, inter alia, a test nucleic acid strand complementary to a target DNA strand, said nucleic acid strand comprises a sufficient number of ribonucleotide residues that span the position of said point mutation to be detected to form a target DNA strand/test nucleic acid strand duplex and RNase H cleavage of said target DNA strand/test nucleic acid strand duplex. Kits and arrays for detecting a point mutation in a DNA strand comprising test nucleic acid strand comprising a sufficient number of ribonucleotide residues that span the position of said point mutation to be detected are also provided.
Nucleic acid hybridization, in the forty years since its discovery, has become a powerful tool with implications for biology, medicine and industry. Hybridization assays are based on the very specific base pairing that is found in hybrids of DNA and RNA. Base sequences of analytical interest appearing along a strand of nucleic acid can be detected very specifically and sensitively by observing the formation of hybrids in the presence of a probe nucleic acid known to comprise a base sequence that is complementary with the sequence of interest. Nucleic acid hybridization has been used for a wide variety of purposes including, for example, identification of specific clones from cDNA and genomic libraries, detecting single base pair polymorphisms in DNA, generating mutations by oligonucleotide mutagenesis, amplifying nucleic acids from single cells or viruses, or detecting microbial infections.
Recent advances in nucleic acid hybridization methods have greatly expanded the scope and extent of its potential applications. Of great interest are approaches to miniaturize hybridization reactions by preparing xe2x80x9cmicroarray biochipsxe2x80x9d (or xe2x80x9cDNA chipsxe2x80x9d) containing large numbers of oligonucleotide probes prepared, for example, through VLSIPS(trademark) technology (See U.S. Pat. Nos. 5,143,854 or 5,561,071). These approaches offer great promise for a wide variety of applications. Microarray biochips are useful for sequencing nucleic acid by hybridization (see, for example, U.S. Pat. No. 5,741,644), for diagnosis of human immunodeficiency virus (see, for example, U.S. Pat. No. 5,861,242) and for screening potential DNA binding drugs (see, for example, U.S. Pat. No. 5,556,752).
When using nucleic acid microarrays, there are two general approaches for detecting hybridization to a nucleic acid. Detection can be accomplished if the target nucleic acid is labeled (xe2x80x9cdirect labeling approachxe2x80x9d). Alternatively, detection can be accomplished by a second probe that is detectably labeled and which can hybridize to the nucleic acid of the sample, which is hybridized to the first probe immobilized on the array (xe2x80x9cindirectxe2x80x9d labeling approach).
Bagwell, U.S. Pat. No. 5,607,834 discloses a fluorescent probe for binding to a polynucleotide target and methods using such fluorescent probes that comprises: an oligonucleotide having a segment complementary to the polynucleotide target, the oligonucleotide forming two imperfect hairpins both of which together include the segment except for one nucleotide; and one donor fluorophore and one acceptor fluorophore covalently attached to the oligonucleotide so that only when the imperfect hairpins are formed, the donor fluorophore and the acceptor fluorophore are in close proximity to allow resonance energy transfer therebetween. The fluorescent probes disclosed in Bagwell must contain xe2x80x9cimperfect hairpins,xe2x80x9d i.e., containing mismatches in the double-stranded stem segment. In addition, Bagwell does not disclose or teach any immobilized arrays of oligonucleotide probes.
Nazarenko et al., U.S. Pat. No. 5,866,336 disclose an oligonucleotide containing a hairpin structure for use as a primer in detecting a target nucleotide sequence. Similar probes are described in Mergny et al., Nucleic Acids Res., 22:920-928 (1994). Blok and Kramer, Molecular and Cellular Probes, 11: 187-194 (1997) describe an amplification RNA probe containing a molecular switch, i.e., a plurality of hairpin structures. Fujiwara and Oishi, Nucleic Acids Res., 26:5728-5733 (1998) describe a method of covalent attachment of probe DNA to double-stranded target DNA where an imperfect hairpin was used to hybridize to a target DNA. Sriprakash and Hartas, Gene Anal. Techn., 6:29-32 (1989) describe a method of generating radioisotope labeled probe with hairpin nucleic acid structure. One common feature of the hairpin structure-containing probes described in the above references is that the nucleotide sequence complementary to a target nucleotide sequence always resides in the single-stranded, not double-stranded, segment of the hairpin structure.
Berkower et al., J. Biol. Chem., 248(17):5914-21 (1973) describe isolation and characterization of an endonuclease, i.e., RNase H, from Escherichia coli specific for ribonucleic acid in ribonucleic acid-deoxyribonucleic acid hybrid structures. Donis-Keller, Nucleic Acids Res., 7(1):179-92 (1979) disclose that the hybridization of a DNA oligonucleotide (a specific tetramer or longer) will direct a cleavage by RNase H (EC 3.1.4.34) to a specific site in RNA. The resulting fragments can then be labeled at their 5xe2x80x2 or 3xe2x80x2 ends, purified, and sequenced directly. This procedure is demonstrated with two RNA molecules of known sequence: 5.8S rRNA from yeast (158 nucleotides) and satellite tobacco necrosis virus (STNV) RNA (1240 nucleotides).
The direct labeling approach can be problematic because nucleic acid labeling methods may fail to label different nucleic acids in a mixture equally. In addition, direct labeling may introduce mutations or other chemical modifications of the sample nucleic acid that prohibit or reduce hybridization.
Detection of hybridization in a microarray biochip by indirect labeling also can be problematic because background hybridization between the second probe may hybridize to the first probe immobilized on the microarray, giving rise to a high false-positive assay background. If the microarray utilizes only a single probe or very limited set of probes, the background may be reduced in the indirect labeling format by designing the specific second probe such that it does not hybridize to the immobilized probes on the array. However, when the microarray contains a wide variety of probe sequences for simultaneously detecting a variety of different nucleic acid targets (the reason for miniaturizing hybridization), designing second probes that are specific and that can avoid background hybridization to the immobilized probes becomes extremely difficult, if not impossible. Accordingly, a need exists for improved hybridization in general and for detecting hybridization and point mutation on microarray formats in particular. The present invention addresses this and other related needs in the art.
In one aspect, the present invention provides a method for detecting a point mutation in a DNA strand, which method comprises: a) hybridizing a target DNA strand containing or suspected of containing a point mutation with a test nucleic acid strand complementary to said DNA strand to form a target DNA strand/test nucleic acid strand duplex, said nucleic acid strand comprising a sufficient number of ribonucleotide residues to span the position of said point mutation to be detected; b) contacting said target DNA strand/test nucleic acid strand duplex formed in step a) with an RNase H; and c) determining whether said ribonucleotide residues within said test nucleic acid strand are cleaved by said RNase H, wherein said ribonucleotide residues within said test nucleic acid strand are cleaved by said RNase H in the absence of mismatch at said position of said point mutation and said ribonucleotide residues within said test nucleic acid strand are not cleaved by said RNase H in the presence of mismatch at said position of said point mutation and the presence or absence of a point mutation in said target DNA is assessed.
Any suitable nucleic acid strand comprising a sufficient number of ribonucleotide residues that span the position of said point mutation to be detected can be used in the present methods. The test nucleic acid strand can comprise ribonucleotide residues only. Alternatively, the test nucleic acid strand can comprise both ribonucleotide residues and deoxyribonucleotide residues. The test nucleic acid strand can also comprise ribonucleotide residues, deoxyribonucleotide residues and peptide bonds or linkages. In a specific embodiment, the test nucleic acid strand comprises ribonucleotide residues, deoxyribonucleotide residues and peptide bonds or linkages beyond the sufficient number of ribonucleotide residues that span the position of said point mutation to be detected.
Any suitable nucleic acid strand, whether linear and/or circular, can be used in the present methods. In a specific embodiment, the test nucleic acid strand is a part of a hairpin probe having a loop and a stem regions, wherein the loop region has more than 2 nucleotide residues and the target DNA strand and the test nucleic acid strand are hybridized under conditions that favor intermolecular hybridization between the target DNA strand and the test nucleic acid strand over intramolecular hybridization of the test nucleic acid strand itself. The sufficient number of ribonucleotide residues that span the position of said point mutation to be detected can be located within the loop or stem region of the hairpin probe. Preferably, the sufficient number of ribonucleotide residues that span the position of said point mutation to be detected is located within the loop and stem regions of the hairpin probe.
The hairpin probe can further comprise an element or a modification that facilitates intramolecular crosslinking of the test nucleic acid strand upon suitable treatment. The element can be a chemically or photoactively activatable crosslinking agent, e.g., a furocoumarin. The element can also be a macromolecule having multiple ligand binding sites, e.g., a component of biotin-avidin binding system.
When hairpin probes are used in the present method, the conditions that favor intermolecular hybridization between the target DNA strand and the test nucleic acid strand over intramolecular hybridization of the test nucleic acid strand itself can be achieved by any suitable methods, e.g., by controlling compositions of the target DNA strand and the test nucleic acid strand so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid. Preferably, the Tm of the intermolecular hybrid is at least 2xc2x0 C. higher than the Tm of the intramolecular hybrid.
In a preferred embodiment, the sufficient number of ribonucleotide residues within the test nucleic acid strand can comprise at least a ribonucleotide sequence having the formula 5xe2x80x2-RXR-3xe2x80x2, 5xe2x80x2-RRX-3xe2x80x2 or 5xe2x80x2-RRXR-3xe2x80x2, or a complementary strand thereof, wherein R is an ribonucleotide residue complementary to its corresponding deoxyribonucleotide in said target DNA strand and X represents the position of said point mutation to be detected and X is a ribonucleotide residue that is complementary or not complementary to its corresponding deoxyribonucleotide in said target DNA strand. In a specific embodiment, X in the above formulas can be complementary to the corresponding deoxyribonucleotide that would be present in a wild-type target DNA strand and cleavage of the ribonucleotide residues within the test nucleic acid strand indicates the absence of a point mutation at position X and failure of the cleavage of the ribonucleotide residues within the test nucleic acid strand indicates the presence of a point mutation at position X. In another specific embodiment, X is not complementary to the corresponding deoxyribonucleotide that would be present in a wild-type target DNA strand and the cleavage of the ribonucleotide residues within the test nucleic acid strand indicates the presence of a point mutation at position X.
The cleavage of the ribonucleotide residues by RNase H can be assessed by suitable methods. For example, the cleavage of the ribonucleotide residues can be assessed by analyzing the disappearance of the target DNA strand/test nucleic acid strand duplex, e.g., by gel electrophoresis. Any type of gel electrophoresis, including agarose gel electrophoresis, pulsed-field gel electrophoresis, capillary electrophoresis and polyacrylamide gel electrophoresis, can be used (See generally, Ausubel (Ed.) Current Protocols in Molecular Biology, 2. Preparation and Analysis of DNA and 4. Preparation and Analysis of RNA, John Wiley and Sons, Inc. (2000)). Other suitable analytical methods such as mass spectrometry, chromatograph, filtration and centrifugation can also be used. In a preferred embodiment, each of the target DNA strand and the test nucleic acid strand contains an element, whereby the formation of the target DNA strand/test nucleic acid strand duplex brings the two elements into close proximity to generate a detectable signal, and the cleavage of the ribonucleotide residues disrupts or interferes with the close proximity of the two elements and alters the detectable signal. More preferably, the elements belong to a enzyme/substrate pair or are components of a fluorescence resonance energy transfer (FRET) system.
The present method can be conducted in a liquid or solution. Alternatively, the present method can be conducted on a surface, e.g., by using a test nucleic acid strand that is immobilized on a solid support.
The present method can be conducted to detect a single point mutation at a time. Preferably, the present method can be conducted in high throughput mode, i.e., by analyzing a plurality of point mutations simultaneously. For example, a plurality of the test nucleic acid strands immobilized on a solid support can be used. Preferably, each of the plurality of the test nucleic acid strands is capable of detecting a different point mutation. More preferably, a plurality of samples is assayed simultaneously using a plurality of the test nucleic acid strands, either in a liquid or solution, or on a surface, wherein each of the plurality of the test nucleic acid strands is capable of detecting a different point mutation.
In another aspect, the present invention provides a kit for detecting a point mutation in a DNA strand, which kit comprises: a) a test nucleic acid strand that is complementary to a target DNA strand containing or suspected of containing a point mutation to be detected and capable of forming a target DNA strand/test nucleic acid strand duplex, said test nucleic acid strand comprising a sufficient number of ribonucleotide residues to span the position of said point mutation to be detected; and b) an RNase H.
In still another aspect, the present invention provides an array of test nucleic acid strands immobilized on a solid support for detecting a point mutation in a DNA strand, comprising a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of test nucleic acid strands, at least one of the test nucleic acid strands comprising a sufficient number of ribonucleotide residues to span the position of said point mutation to be detected to form a target DNA strand/test nucleic acid strand duplex.